Ex vivo engineering of patient-derived regulatory T (Treg) cells holds promise as a safe and effective approach to preventing graft versus host disease and treating a range of autoimmune diseases, such as type 1 diabetes and multiple sclerosis. However, the methods currently employed to engineer patient-derived cells for thera- peutic use (viral delivery and electroporation) have yet to be optimized to increase the broad availability of per- sonalized immunotherapies to patients in need. This proposal is focused on meeting this need for an across- the-board Treg cell engineering method, from development and optimization in the laboratory to the commer- cial production of personalized Treg cell immunotherapies for prescription use. Microfluidic vortex shedding (VS) is a safe and rapid approach to genetically modify patient-derived CD3+ T cells. VS technology takes advantage of naturally occurring fluid dynamic properties to gently and temporarily porate cell membranes, thereby enabling a rapid, yet safe, approach to T cell transfection that cannot be achieved by current ap- proaches. The objective of this work is to expand the utility of VS to the unique Treg cell population, thus demonstrating the feasibility of VS to develop and manufacture engineered Tregs for cell-based immunother- apies. The research and development objectives are to (1) demonstrate the technical performance of VS-me- diated transfection of human Treg cells with a chimeric antigen receptor (CAR) construct that targets cells ex- pressing the human leukocyte antigen A2, thus indicating clinical utility as a therapeutic treatment to prevent graft versus host disease, and (2) demonstrate the functionality and safety of transfected Treg cells generated by VS in cell-based and in vivo assays. Pending the successful completion of these objectives, CAR-Treg cells will be engineered using patient-derived Treg cells, and commercial-scale processing and enrichment of sufficient genetically modified viable cells for clinical applications will be demonstrated.